Method and kit for immunological detection of Mycobacterium tuberculosis

ABSTRACT

Definitive diagnosis and early start of treatment cannot be made for tuberculosis since conventional methods for detecting a  Mycobacterium tuberculosis  complex require a long time plus enormous labor and expense. Because detection is difficult to perform directly from a biological sample, if the biological sample contains no or a very small amount of MPT64, there is a risk infection with the  Mycobacterium tuberculosis  complex will be missed. The present method and a kit address these long-standing needs by more rapidly and conveniently detecting a  Mycobacterium tuberculosis  complex, without culturing a biological sample containing the  Mycobacterium tuberculosis  complex, in which a biological sample is subjected to a heat-treatment so as to extracellularly secrete a  Mycobacterium tuberculosis  complex-specific secretory protein, particularly, MPB64, and subjecting the resulting treated sample to an immunological measurement/assay.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a U.S. National Stage Application filed under 35U.S.C. § 371 of International Application PCT/JP2013/060301, filed Apr.4, 2013, designating the United States, which claims priority fromJapanese Patent Application 2012-086566, filed Apr. 5, 2012, thecomplete disclosures of which are hereby incorporated herein byreference in their entirety for all purposes.

TECHNICAL FIELD

The present invention relates to a highly safe method and a kit forimmunological detection of a Mycobacterium tuberculosis complex whichcan be conveniently and rapidly performed directly from a biologicalsample containing the Mycobacterium tuberculosis complex without cultureoperation by heat-treating the biological sample at a predeterminedtemperature and immunologically detecting a Mycobacterium tuberculosiscomplex-specific secretory protein thus extracellularly secreted.

BACKGROUND ART

Tuberculosis is currently an infectious disease of high importance,which is said to be killing thousands of people in Japan and millions ofpeople worldwide every year. In Japan, a person suspected of havingtuberculosis infection must be hospitalized according to the InfectiousDisease Law. A physician must immediately report to the authority whenhaving diagnosed a patient with tuberculosis, and thus prompt responsesare required.

Mycobacterium tuberculosis (human type of tuberculosis bacterium),Mycobacterium bovis (bovine type of tuberculosis bacterium),Mycobacterium microti (murine type of tuberculosis bacterium), andMycobacterium africanum (African type of tuberculosis bacterium), whichbelong to a Mycobacterium tuberculosis complex, are known as acid-fastbacteria pathogenic to humans. Mycobacterium avium, Mycobacteriumkansasii, Mycobacterium marinum, and the like are known asnon-tuberculous mycobacteria.

Human infection with the Mycobacterium tuberculosis complex is mainlycaused by Mycobacterium tuberculosis and, in some rare cases, byMycobacterium bovis. Cases of infection with Mycobacterium avium, amongthe non-tuberculous mycobacteria, have been increased in recent years.Since both of these infections present similar clinical symptoms, theidentification of a causative microbe is also important for determiningcourses of treatment.

Thus, the differential detection of tuberculosis infection ornon-tuberculous mycobacterial infection is also important for reducingburdens on patients and appropriately conducting early treatment. Inanother aspect, the differential detection is also important for thepurpose of preventing a third person from having secondary infectionfrom the public health standpoint.

Heretofore, detection methods based on culture have been practiced asmethods for detecting the Mycobacterium tuberculosis complex. Suchculture methods are performed by inoculating a biological sample to betested to either a liquid medium or a solid medium and detecting grownbacterial cells. The method using the liquid medium carries a high riskof secondary infection during operation and thus requires a highly safefacility and apparatus, though this method can shorten the cultureperiod of the biological sample to be tested. The method using the solidmedium requires a period as long as approximately 2 months to obtaindetection results. The Mycobacterium tuberculosis complex must becultured using a highly safe apparatus in a highly safe environment withgreat care being taken for preventing secondary infection.

As a method for identifying the Mycobacterium tuberculosis complex fromthe culture of the above-cultured biological sample, a convenientdetection method is known which involves immunologically assaying aMycobacterium tuberculosis complex-specific protein secreted into themedium.

Japanese Patent Laid-Open No. 11-108931 discloses a method for detectingthe presence of a Mycobacterium tuberculosis complex, comprising:inoculating a biological sample collected from a tuberculosis patient,to a solid medium or a liquid medium; culturing the Mycobacteriumtuberculosis complex for a few days to a few weeks; and thenimmunologically detecting a Mycobacterium tuberculosis complex-specificsecretory protein MPB64 secreted into the medium. According to thisreference, this method permits identification and detection of theMycobacterium tuberculosis complex immediately after the culture andreduces the risk of secondary infection during operation, compared withconventional methods.

This method, however, involves culturing the Mycobacterium tuberculosiscomplex contained in the biological sample and using the resultingcultures as a sample. Hence, the method requires almost the same amountof time as the culture period until the Mycobacterium tuberculosiscomplex is detected and identified, and thus requires enormous labor andexpense.

As described in Japanese Patent Laid-Open No. 11-108931, MPB64 is aMycobacterium tuberculosis complex-specific secretory protein which isproduced by Mycobacterium bovis BCG (M. bovis BCG) and extracellularlysecreted. MPT64 is a Mycobacterium tuberculosis complex-specificsecretory protein which is produced by Mycobacterium tuberculosis andextracellularly secreted.

MPB64 and MPT64 are known as identical substances. They may beindividually referred to elsewhere in describing the inventions herein.They may also be conveniently designated as MPB64/MPT64 elsewhereherein.

Japanese Patent Laid-Open No. 2002-62299 states that the Mycobacteriumtuberculosis complex-specific secretory protein MPT64 can be detected byimmunological assay without culturing a Mycobacterium tuberculosiscomplex contained in a biological sample. This method involves treatinga biological sample such as sputum and detecting MPT64 contained in thebiological sample so that the presence of the Mycobacterium tuberculosiscomplex is detected.

However, even if the Mycobacterium tuberculosis complex is present inthe biological sample, this method cannot detect the Mycobacteriumtuberculosis complex unless MPB64 is extracellularly secreted by theMycobacterium tuberculosis complex and contained in the biologicalsample. In addition, a small abundance of MPT64, albeit secreted,requires a large amount of the biological sample and complicates theoperation. This poses increased burdens on test subjects and persons incharge of the tests. At the same time, infection with the Mycobacteriumtuberculosis complex might be missed, resulting in public healthproblems.

International Publication No. WO 2009/084481 discloses a method fordiagnosing tuberculosis infection more rapidly and more safely withhigher accuracy than ever, comprising specifically detecting MPB64 in abiological sample using an antibody against a particular epitope inMPB64. According to this method, an applicable sample can be a culturesobtained by culturing a biological sample using a small amount of aliquid medium only for a time before bacterial cells of theMycobacterium tuberculosis complex substantially start to grow.

This method, however, still demands a method for promoting the secretionof MPB64 from the bacterial cells before growth so that a larger amountof MPB64 accumulates in the medium.

CONVENTIONAL TECHNICAL DOCUMENTS Patent Documents

-   Patent Document 1: Japanese Patent Laid-Open No. 11-108931-   Patent Document 2: Japanese Patent Laid-Open No. 2002-62299-   Patent Document 3: International Publication No. WO 2009/084481

SUMMARY OF INVENTION Problems to be Solved by the Invention

As described above, the conventional methods require a long time fordetecting a Mycobacterium tuberculosis complex and also require enormouslabor and expense. For these reasons, definitive diagnosis and earlystart of treatment cannot be made for tuberculosis. Thus, there has beena demand for a more rapid method. Also, the direct detection from abiological sample cannot be performed if the biological sample containsno or a very small amount of MPB64/MPT64. As a result, infection withthe Mycobacterium tuberculosis complex might be missed. Thus, there hasbeen a need for a more reliable detection method. An object of thepresent invention is to solve these problems by providing a convenient,rapid, and more reliable method for detecting a Mycobacteriumtuberculosis complex.

Means for Solving the Problems

The present inventors have conducted diligent studies to solve the aboveproblems and consequently found that the problems can be solved byheat-treating a biological sample containing a Mycobacteriumtuberculosis complex so as to extracellularly secrete a Mycobacteriumtuberculosis complex-specific secretory protein such as MPB64 anddetecting the thus-secreted protein, and as a result, the presentinvention has been completed.

According to one aspect, the present invention provides a method fordetecting a Mycobacterium tuberculosis complex, comprising subjecting aMycobacterium tuberculosis complex-specific protein to an immunologicalassay, wherein said protein is extracellularly secreted by subjecting abiological sample containing the Mycobacterium tuberculosis complex to aheat-treatment. According to another aspect, the present invention alsoprovides a kit for detection of a Mycobacterium tuberculosis complex,comprising at least a treatment container in which a biological samplecontaining the Mycobacterium tuberculosis complex is subjected to aheat-treatment, and an immunological assay apparatus for detecting aMycobacterium tuberculosis complex-specific secretory protein secretedby the heat-treatment, as a kit for detection of a Mycobacteriumtuberculosis complex for the purpose of carrying out the above method.The kit may further comprise a sample treating agent and/or a solvent.The detection method and the kit of the present invention are capable ofrapidly and conveniently detecting the Mycobacterium tuberculosiscomplex. According to a further aspect, the present invention alsoprovides a method for extracellularly secreting a Mycobacteriumtuberculosis complex-specific protein, comprising subjecting abiological sample containing the Mycobacterium tuberculosis complex to aheat-treatment.

BRIEF EXPLANATION OF DRAWINGS

FIG. 1 is a graph showing the relationship between the heat-treatmenttemperature and the effect of promoting extracellular MPB64 secretion,when bacterial cells of a M. bovis BCG Tokyo strain were used.

FIG. 2 is a graph showing the relationship between the heat treatmenttime and the effect of promoting extracellular MPB64 secretion, whenbacterial cells of a M. bovis BCG Tokyo strain were used.

DESCRIPTION OF EMBODIMENTS

The biological sample used in the present invention is not particularlylimited as long as the biological sample may contain a Mycobacteriumtuberculosis complex. Examples of the biological sample include sputum,pleural effusion, bronchial secretion, gastric juice, blood, spinalfluid, urine, and feces. Preferably, sputum is used because of its highbacterial concentration. Alternatively, bronchial lavage fluid collectedduring respiratory examination, a tissue collected from the bronchus orthe lung, or the like may be used as the biological sample. Thesebiological samples may each be used alone or may be used as a mixture oftwo or more thereof.

The heat-treatment of the biological sample is preferably carried out byplacing the biological sample in a hermetically sealable container andthen subjecting the container as a whole to a heat-treatment because thebiological sample to be treated contains a highly infectiousMycobacterium tuberculosis complex. The heat-treatment operation ispreferably performed within a safety cabinet from the viewpoint ofsecuring the safety of an operator. The container for use in theheat-treatment is not particularly limited as long as the container canmaintain its hermetically sealed state and can resist a predeterminedheating temperature. The container can be appropriately selectedaccording to the biological sample to be treated. The container may beequipped, at its opening, with a dropper nozzle with a filter so as tofacilitate the discharge of the thus-treated biological sample from thecontainer, for immunological assay. Use of the dropper nozzle with afilter is preferred because the dropper nozzle with a filter can removesolid matter such as denatured components contained in the treatedbiological sample and further, can conveniently put drops of theresulting biological sample to an immunological assay apparatus.

The biological sample may be subjected to a treatment such as lysis witha sample treating agent according to its properties before theheat-treatment. For example, when sputum is used as the sample, it maybe lysed with an alkaline substance, a reducing substance, a protease, asugar, a surfactant, a protein denaturant, or the like, which reducesthe viscosity of sputum, in order to improve the efficiency of theheat-treatment. Particularly, a treatment using sodium hydroxide as analkaline substance and N-acetyl-L-cysteine as a reducing substance incombination is convenient and is preferably used.

According to another embodiment, the biological sample may be dispersedor dissolved in a solvent and then subjected to a heat-treatment. Thesolvent can be, for example, any solvent that can keep the Mycobacteriumtuberculosis complex contained in the biological sample alive. A buffersolution such as phosphate-buffered saline or a liquid medium for use inacid-fast bacterium isolation culture such as Middlebrook 7H9 can beused. Particularly, a liquid medium is preferably used because theprotein needs to be extracellularly secreted by the heat-treatmentwithout impairing the activity of the Mycobacterium tuberculosis complexand because the thus-treated sample can be subjected directly to animmunological assay. Alternatively, the biological sample lysed with thesample treating agent may be redispersed in the solvent.

The heating temperature for the biological sample can be any temperatureat which the protein can be extracellularly secreted into the biologicalsample or the solvent by the Mycobacterium tuberculosis complex, and ispreferably in the range of 40° C. to 60° C., more preferably in therange of 40° C. to 55° C. Particularly, the temperature that effectivelypromotes the secretion is in the range of 45° C. to 50° C.

The treatment time for the biological sample can be a time that issufficient to extracellularly secrete a detectable amount of theMycobacterium tuberculosis complex-specific secretory protein while thetreatment temperature is kept within the above range. The treatment timeis preferably 15 minutes or longer, more preferably 15 minutes or longerand 2.5 hours or shorter, further preferably 15 minutes or longer and1.5 hours or shorter, most preferably 30 minutes or longer and 1 hour orshorter.

The sample thus heat-treated may be subjected to the inactivationtreatment of the Mycobacterium tuberculosis complex merely by raisingthe heating temperature. The temperature for the inactivation treatmentof the Mycobacterium tuberculosis complex is not particularly limitedbut is preferably 100° C. The inactivation of the Mycobacteriumtuberculosis complex contained in the sample allows immunological assayto be conducted safely without the risk of secondary infection.

The heating apparatus for use in the heat-treatment is not particularlylimited as long as the apparatus is capable of keeping the temperatureconstant. A thermostat bath, a heater block, an incubator, or the likecan be used. A small heating apparatus is particularly preferred becauseall the procedures for detecting the Mycobacterium tuberculosis complexcan be completed within a safety cabinet.

According to the present invention, the Mycobacterium tuberculosiscomplex-specific protein can be extracellularly secreted by theheat-treatment of the biological sample. The presence of theMycobacterium tuberculosis complex can therefore be easily detected byimmunological assay or the like using the biological sample thustreated.

In the present invention, the Mycobacterium tuberculosiscomplex-specific protein to be extracellularly secreted is notparticularly limited as long as the protein is extracellularly secreted.Examples of the protein that is extracellularly secreted by theMycobacterium tuberculosis complex include many proteins such as MPB64(i.e., MPT64), MPB70, ESAT-6, and CFP-10. Of these proteins, a proteinthat is extracellularly secreted in a large amount in a short time ispreferred as a protein used as an index for indicating the presence ofthe Mycobacterium tuberculosis complex in the immunological assay usedin the present invention. For example, MPB64, i.e., MPT64, isextracellularly secreted or released in a large amount by theheat-treatment of the biological sample according to the presentinvention without culture, i.e., before the bacterial cells start togrow. From such a viewpoint, MPB64, i.e., MPT64 is preferably used as atarget for the detection of the presence of the Mycobacteriumtuberculosis complex. The extracellularly secreted MPB64, i.e., MPT64can be immunologically assayed so as to determine the presence orabsence of the Mycobacterium tuberculosis complex.

In the detection method of the present invention, the immunologicalassay is not particularly limited and is preferably a sandwichimmunoassay using a first antibody and a second antibody against theMycobacterium tuberculosis complex-specific secretory protein,particularly, enzyme-linked immunosorbent assay (ELISA) orimmunochromatographic assay. The first antibody and the second antibodymay be the same or may be different from each other as long as theypermit detection of the Mycobacterium tuberculosis complex-specificsecretory protein.

This immunological assay can be any method capable of immunologicallydetecting MPB64 extracellularly secreted by the heat-treatment of thebiological sample and is preferably immunochromatographic assay,particularly preferably immunochromatographic assay using an anti-MPB64monoclonal antibody. The immunochromatographic assay for MPB64 detectioncan be easily conducted in accordance with the method described inJapanese Patent Laid-Open No. 11-108931. In addition, Capilia® TB(manufactured by TAUNS Laboratories, Inc.), a commercially availableimmunochromatographic assay apparatus for detection of MPB64, i.e.,MPT64, can be used.

EXAMPLES

The present invention will be described further specifically by way ofExamples below. However, the present invention is not intended to belimited by these Examples.

Reference Example 1 Preparation of a Bacterial Solution to be Tested

4.7 g of Middlebrook 7H9 broth base (manufactured by Difco LaboratoriesInc.) was dissolved in 900 ml of distilled water containing 0.5 g ofTween 80. The solution was high-pressure sterilized in an autoclave at121° C. for 10 minutes. After cooling, 100 ml of ADC Enrichment(albumin-dextrose-catalase) was aseptically added thereto to prepare aMiddlebrook 7H9 liquid medium.

A M. bovis BCG Tokyo strain (hereinafter, referred to as a BCG strain)was inoculated to the above-prepared liquid medium, and culturedaccording to a standard method. The culture was continued untilturbidity corresponding to McFarland standards No. 1 was obtained. Theresulting bacterial solution was centrifuged to recover bacterial cells.The recovered bacterial cells were resuspended by the addition of theabove-mentioned Middlebrook 7H9 liquid medium and then furthercentrifuged to recover bacterial cells. This operation was repeatedthree times. The bacterial cells were washed to remove Mycobacteriumtuberculosis complex secretory proteins attached to the bacterial cells.100 μL of the supernatant after the washing operation was applied toCapilia® TB (manufactured by TAUNS Laboratories, Inc.), animmunochromatographic assay apparatus for MPB64 detection. As a result,this product exhibited negativity to confirm that secreted MPB64 wasabsent in the supernatant.

Example 1 Study on Optimum Condition for Heat-Treatment Temperature

The optical density of the bacterial solution having the turbiditycorresponding to McFarland standards No. 1, prepared in ReferenceExample 1, was adjusted to O.D. 0.1 at an absorbance of 530 nm toprepare a bacterial solution for testing (bacterial count: correspondingto 10⁷ CFU). The bacterial solution for testing was further diluted witha liquid medium to prepare a 10-fold diluted bacterial solution(bacterial count: corresponding to 10⁶ CFU) and a 100-fold dilutedbacterial solution (bacterial count: corresponding to 10⁵ CFU). 200 μlof each prepared bacterial solution was dispensed to each plastic tube(a tube for nucleic acid amplification) and heat-treated using atemperature-controllable heat block for a nucleic acid amplificationapparatus. The heating temperature was set to temperatures differing by5° C. from 35° C. to 75° C. The heat-treatment was carried out for 30minutes. Samples heat-treated at an ordinary culture temperature of 35°C. were used as controls. A 100 μl aliquot was collected from eachsample thus treated, and applied to Capilia® TB (manufactured by TAUNSLaboratories, Inc.) in the same way as in Reference Example 1 to detectthe presence of MPB64. The absorbance of the reading area was measuredusing Immno Chromato-Reader (manufactured by Otsuka Electronics Co.,Ltd.).

The results are shown in FIG. 1. In FIG. 1, the ordinate represents themeasurement value of color intensity (absorbance) in Capilia® TB(manufactured by TAUNS Laboratories, Inc.), and the abscissa representsthe heating temperature. The relationship between the absorbance and thetreatment temperature is indicated by solid line for the results aboutthe bacterial solution for testing (bacterial count: corresponding to10⁷ CFU), by broken line for the results about the 10-fold dilutedbacterial solution (bacterial count: corresponding to 10⁶ CFU), and bylong broken line for the results about the 100-fold diluted bacterialsolution (bacterial count: corresponding to 10⁵ CFU).

As seen from the results, all of the tested samples exhibited a rise inabsorbance at 40° C. to 60° C., particularly, a marked rise at 45° C. to50° C. This indicates that the heat-treatment of the bacterial cellspromoted their extracellular MPB64 secretion. The controls treated underan ordinary culture condition of 35° C. exhibited no rise in absorbance,demonstrating that just keeping a sample at room temperature or underconditions equivalent thereto does not cause MPB64 to be extracellularlysecreted. On the other hand, no rise in absorbance was confirmed in atemperature range higher than the temperature range from 40° C. to 60°C. where the secretion of MPB64 was confirmed. This showed that MPB64 isnot secreted in a temperature range equal to or higher than 60° C.

Accordingly, from these results, the heat-treatment of a bacterialsample at 40° C. to 60° C., particularly, at 45° C. to 50° C. wasconfirmed to markedly promote extracellular MPB64 secretion and markedlyincrease the detection rate of immunological assay.

Example 2 Study on Optimum Condition for Heat-Treatment Temperature

The optical density of the bacterial solution having the turbiditycorresponding to McFarland standards No. 1, prepared in ReferenceExample 1, was adjusted to O.D. 0.1 at an absorbance of 530 nm toprepare a bacterial solution for testing (bacterial count: correspondingto 10⁷ CFU). 200 μl of the prepared bacterial solution was dispensed toeach plastic tube (a tube for nucleic acid amplification) andheat-treated using a temperature-controllable heat block for a nucleicacid amplification apparatus. The heating temperature was set to 35° C.,13 temperatures differing by 2° C. from 40° C. to 64° C., and 2temperatures of 70° C. and 80° C. as a high temperature range. Theheat-treatment was carried out for 60 minutes. Samples heated-treated atan ordinary culture temperature of 35° C. were used as controls. A 100μl aliquot was collected from each sample thus treated, and applied toCapilia® TB (manufactured by TAUNS Laboratories, Inc.) in the same wayas in Reference Example 1 to detect the presence of MPB64. Evaluationwas conducted by the visual observation of the color intensity of alabeling substance accumulated in the reading area. The color intensitywas visually evaluated as the degree of magenta color on the basis of 5grades: − (not colored), ± (weakly colored), + (distinctly colored), ++(more distinctly colored), and +++ (markedly colored). The evaluationresults are shown in Table 1.

TABLE 1 Treatment temperature Evaluation 35° C. (control) − 40° C. ± 42°C. + 44° C. + 46° C. ++ 48° C. +++ 50° C. +++ 52° C. ++ 54° C. + 56°C. + 58° C. + 60° C. ± 62° C. − 64° C. − 70° C. − 80° C. −

As seen from the results of Table 1, a rise in color intensity wasexhibited at 40° C. to 60° C., and particularly, a marked rise wasconfirmed at 44° C. to 52° C. This indicates that the heat-treatment ofthe bacterial cells promoted their extracellular MPB64 secretion, as inExample 1. The controls treated under an ordinary culture condition of35° C. exhibited no rise in absorbance, demonstrating that just keepinga sample at room temperature or under conditions equivalent thereto doesnot cause MPB64 to be extracellularly secreted. On the other hand, norise in absorbance was confirmed at the temperatures equal to or higherthan 62° C. or in the high temperature range of 70° C. and 80° C. Thisshowed that MPB64 is not secreted in a temperature range equal to orhigher than 60° C.

Accordingly, from these results, the heat-treatment of a bacterialsample at 40° C. to 60° C., particularly, at 45° C. to 50° C. wasconfirmed to markedly promote extracellular MPB64 secretion and markedlyincrease the detection rate of immunological assay.

Example 3 Study on Optimum Condition for Heat Treatment Time

The optical density of the bacterial solution having the turbiditycorresponding to McFarland standards No. 1, prepared in ReferenceExample 1, was adjusted to O.D. 0.1 at an absorbance of 530 nm toprepare a bacterial solution for testing (bacterial count: correspondingto 10⁷ CFU). 200 μl of the prepared bacterial solution was dispensed toeach plastic tube and heat-treated using a temperature-controllable heatblock for a nucleic acid amplification apparatus. The heat operation wascarried out with the heating temperature set to temperatures differingby 5° C. from 35° C. to 75° C. The treatment time was set to 0 minutes,15 minutes, 30 minutes, and 60 minutes. A 100 μl aliquot was collectedfrom each sample thus treated, and applied to Capilia® TB (manufacturedby TAUNS Laboratories, Inc.) in the same way as in Example 1. Theabsorbance of the reading area was measured using Immno Chromato-Reader(manufactured by Otsuka Electronics Co., Ltd.).

The results are shown in FIG. 2. In FIG. 2, the ordinate represents themeasurement value of color intensity (absorbance) in Capilia® TB(manufactured by TAUNS Laboratories, Inc.), and the abscissa representsthe heating temperature. The relationship between the absorbance and thetreatment temperature is indicated by broken line for the results aboutthe samples treated for the treatment time of 15 minutes, by solid linefor the results about the samples treated for the treatment time of 30minutes, and by long broken line for the results about the samplestreated for the treatment time of 60 minutes.

As seen from the results, all of the samples treated for the treatmenttime of 15 minutes, 30 minutes, and 60 minutes exhibited a marked risein absorbance at the temperature of 45° C. to 50° C. No difference wasseen among the treatment times. Even the samples treated for the heatingtime of 60 minutes were not confirmed to largely differ from the othersamples. Accordingly, the heat treatment time of at least 15 minutes wasconfirmed to have the effect of promoting extracellular MPB64 secretion.

Example 4 Study on Optimum Condition for Heat Treatment Time

The optical density of the bacterial solution having the turbiditycorresponding to McFarland standards No. 1, prepared in ReferenceExample 1, was adjusted to O.D. 0.01 at an absorbance of 530 nm toprepare a bacterial solution for testing (bacterial count: correspondingto 10⁶ CFU). 200 μl of the prepared bacterial solution was dispensed toeach plastic tube and heat-treated at 50° C. using atemperature-controllable heat block for a nucleic acid amplificationapparatus. The heat-treatment was carried out with the treatment timeset to 0 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 75minutes, 120 minutes, and 150 minutes. As a control, the same bacterialsolution to be tested as above was left at room temperature withoutheat-treatment. A 100 μl aliquot was collected from each sample thustreated, and applied to Capilia® TB (manufactured by TAUNS Laboratories,Inc.) in the same way as in Example 1. The color intensity of anaccumulated labeling substance was visually observed. The colorintensity was visually evaluated as the degree of magenta color on thebasis of 5 grades: − (not colored), ± (weakly colored), + (distinctlycolored), ++ (more distinctly colored), and +++ (markedly colored). Theevaluation results are shown in Table 2.

TABLE 2 50° C. heat-treatment Control (no heat-treatment) EvaluationEvaluation  0 min − − 15 min + − 30 min + − 45 min ++ − 60 min +++ − 75min +++ − 120 min  +++ − 150 min  +++ −

As seen from the results of Table 2, the non-heat-treated control sampleand the sample treated for the heating time of 0 minutes were confirmedto develop no color in the reading area. The heat-treatment for 15minutes was confirmed to produce color derived from extracellular MPB64secretion. The sample heat-treated for 60 minutes was confirmed todevelop marked color. The samples heat-treated for up to 150 minutesexhibited no further rise in color intensity and were confirmed todevelop marked color at the same level as that of the sampleheat-treated for 60 minutes. From these results, heat-treatment for atleast 15 minutes was confirmed to have the effect of promotingextracellular MPB64 secretion, which peaked at 60 minutes.

Example 5 Study on Solvent

The bacterial solution having the turbidity corresponding to McFarlandstandards No. 1, prepared in Reference Example 1, was prepared intobacterial solutions having a bacterial count corresponding to 10⁷ CFU,10⁶ CFU, or 10⁵ CFU using a Tween 80-containing phosphate buffersolution or a Middlebrook 7H9 liquid medium as a solvent for biologicalsample treatment to prepare test samples. 200 μl of each test sample wasdispensed to each plastic tube. Then, the sample was heat-treated at 50°C. for 30 minutes in a heat block. Then, a 100 μl aliquot was collectedfrom the sample thus treated, and applied to Capilia® TB (manufacturedby TAUNS Laboratories, Inc.) in the same way as in Example 1, followedby evaluation. The evaluation was conducted by the visual observation ofthe color intensity of a labeling substance accumulated in the readingarea. The color intensity was visually evaluated as the degree ofmagenta color on the basis of 5 grades: − (not colored), ± (weaklycolored), + (distinctly colored), ++ (more distinctly colored), and +++(markedly colored). The evaluation results are shown in Table 3.

TABLE 3 Solvent Tween 80- Middlebrook containing phosphate 7H9 liquidbuffer solution medium Test Corresponding + ++ sample to 10⁵ CFU(bacterial Corresponding ++ +++ concen- to 10⁶ CFU tration)Corresponding +++ +++ to 10⁷ CFU

As is evident from the results of Table 3, extracellularly secretedMPB64 can be detected in both cases where the Tween 80-containingphosphate buffer solution is used as a solvent and where the Middlebrook7H9 liquid medium is used as a solvent. Thus, no difference inimmunological assay was confirmed among variations in the composition ofa solvent.

Example 6 Detection from Sputum Sample

The bacterial solution having the turbidity corresponding to McFarlandstandards No. 1, prepared in Reference Example 1, was added to sputumthat had been confirmed to be negative for tuberculosis infection toprepare a pseudo-positive sputum sample having a bacterial countcorresponding to 10⁷ CFU. This sample was dispensed to plastic tubes. Toeach of these sputum samples, N-acetyl-L-cysteine and an aqueous sodiumhydroxide solution were added in equal amounts, and the mixture was leftstanding at room temperature for 15 minutes to lyse the sample. Then,the sample was heat-treated at 50° C. for 30 minutes using a heat block.Then, a 100 μl aliquot was collected from the sample thus treated, andapplied to Capilia® TB (manufactured by TAUNS Laboratories, Inc.) in thesame way as in Example 1, followed by evaluation. Control sputum sampleswere treated in the same way as above except that they were leftstanding at room temperature for 30 minutes without the heat-treatment,followed by evaluation. The evaluation was conducted by the visualobservation of the color intensity of a labeling substance accumulatedin the reading area. The color intensity was visually evaluated as thedegree of magenta color on the basis of 5 grades: − (not colored), ±(weakly colored), + (distinctly colored), ++ (more distinctly colored),and +++ (markedly colored). The evaluation results are shown in Table 4.

TABLE 4 No heat-treatment 50° C. heat-treatment Sample <1> − + Sample<2> − + Sample <3> − to ± ++ Sample <4> − +

As a result, the heat-treated samples were confirmed to develop evidentcolor in the reading area, demonstrating the presence of MPB64 in thesamples. By contrast, the control samples evidently differed from theheat-treated samples, though some samples exhibited slight color in thereading area. Accordingly, the heat-treatment was confirmed to promoteMPB64 secretion even in a sputum sample, demonstrating that theMycobacterium tuberculosis complex can be detected rapidly andconveniently from a biological sample.

INDUSTRIAL APPLICABILITY

The present invention provides a method for detecting a Mycobacteriumtuberculosis complex which can be conveniently and rapidly performeddirectly from a biological sample containing the Mycobacteriumtuberculosis complex without culture operation by heat-treating thebiological sample at a predetermined temperature and immunologicallyassaying a Mycobacterium tuberculosis complex-specific secretory proteinthus extracellularly secreted, and also provides a kit directed thereto.The present invention is useful not only for the detection of theMycobacterium tuberculosis complex but for the appropriate diagnosis andtreatment of tuberculosis.

The invention claimed is:
 1. A method for detecting the presence of aMycobacterium tuberculosis complex-specific protein in a heat-treatedbiological sample, the method comprising subjecting a heat-treatedbiological sample to an immunological assay for the presence of theMycobacterium tuberculosis complex-specific protein, wherein theMycobacterium tuberculosis complex-specific protein is MPB64/MPT64,wherein the heat-treated biological sample to be immunologically assayedis obtained by subjecting a biological sample that may containMycobacterium tuberculosis complex to a heat-treatment carried out at45° C. to 60° C. for a time sufficient to promote extracellularsecretion of the Mycobacterium tuberculosis complex-specific proteinMPB64/MPT64, and wherein the biological sample is lysed with a sampletreating agent before being subjected to the heat-treatment.
 2. A methodaccording to claim 1, wherein the heat-treatment of the biologicalsample is carried out at 45° C. to 55° C.
 3. A method according to claim2, wherein the heat-treatment of the biological sample is carried out at45° C. to 50° C.
 4. A method according to claim 1, wherein thebiological sample is selected from the group consisting of sputum,bronchial secretion, pleural effusion, gastric juice, blood, spinalfluid, urine, feces, bronchial lavage fluid, and a tissue collected fromthe bronchus or the lung.
 5. A method according to claim 4, wherein thebiological sample is sputum.
 6. A method for according to claim 1,wherein the biological sample lysed with a sample treating agent isdispersed or dissolved in a solvent before being subjected to theheat-treatment.
 7. A method according to claim 1, wherein theimmunological assay is a sandwich immunoassay using a first antibody anda second antibody against the Mycobacterium tuberculosiscomplex-specific protein.
 8. A method according to claim 7, wherein theimmunological assay is an immunochromatographic assay.
 9. A methodaccording to claim 7, wherein the immunological assay is ELISA.
 10. Amethod for extracellularly secreting a Mycobacterium tuberculosiscomplex-specific protein MPB64/MPT64, comprising subjecting a biologicalsample containing a Mycobacterium tuberculosis complex that secretes aMycobacterium tuberculosis complex-specific protein MPB64/MPT64 to aheat-treatment that is carried out at 45° C. to 60° C. for 15 to 60minutes whereby said Mycobacterium tuberculosis complex-specific proteinMPB64/MPT64 is extracellularly secreted, wherein the biological sampleis lysed with a sample treating agent before being subjected to theheat-treatment.
 11. A method according to claim 1, said methodcomprising: subjecting the lysed biological sample to the heat-treatmentat 45° C. to 60° C. for 15 to 60 minutes to promote extracellularsecretion of the Mycobacterium tuberculosis complex-specific proteinMPB64/MPT64; preparing the heat-treated sample for immunologicalassaying; and subjecting the heat-treated sample to the immunologicalassay for the presence of Mycobacterium tuberculosis complex-specificprotein MPB64/MPT64.
 12. A method for detecting the presence of aMycobacterium tuberculosis complex-specific protein in a heat-treatedbiological sample, the method comprising subjecting a heat-treatedbiological sample to an immunological assay for the presence of theMycobacterium tuberculosis complex-specific protein, wherein theMycobacterium tuberculosis complex-specific protein is MPB64/MPT64,wherein the heat-treated biological sample to be immunologically assayedfor the presence of the Mycobacterium tuberculosis complex-specificprotein is obtained from a biological sample, which was obtained from asubject, that has been subjected to a heat-treatment carried out at 45°C. to 60° C. for a time sufficient for promoting Mycobacteriumtuberculosis complex that may be present in the biological sample toextracellularly secrete a detectable amount of the Mycobacteriumtuberculosis complex-specific protein MPB64/MPT64, and wherein saidbiological sample has been lysed with a sample treating agent beforebeing subjected to the heat-treatment.
 13. A method for detecting forthe presence of Mycobacterium tuberculosis complex-specific proteinMPB64/MPT64 in a heat-treated biological sample, the method comprising:subjecting an aliquot of a heat-treated biological sample to animmunological assay to detect for the presence of Mycobacteriumtuberculosis complex-specific protein MPB64/MPT64, wherein the aliquotof the heat-treated biological sample to be immunologically assayed forthe presence of said Mycobacterium tuberculosis complex-specific proteinMPB64/MPT64 is obtained by collecting a biological sample from asubject, wherein said biological sample may contain Mycobacteriumtuberculosis complex and said biological sample is selected from thegroup consisting of sputum, bronchial secretion, pleural effusion,gastric juice, blood, spinal fluid, urine, feces, bronchial lavagefluid, and a tissue collected from the bronchus or the lung, lysing thebiological sample with a sample treating agent, subjecting the lysedbiological sample to a heat-treatment carried out at 45° C. to 60° C.for a time sufficient for inducing Mycobacterium tuberculosis complexthat may be present in the biological sample to extracellularly secreteMycobacterium tuberculosis complex-specific protein MPB64/MPT64, andcollecting and preparing the aliquot from the heat-treated biologicalsample.
 14. A method for detecting the presence of Mycobacteriumtuberculosis complex-specific protein MPB64/MPT64 in a biologicalsample, the method comprising: collecting a biological sample from asubject, wherein said biological sample may contain Mycobacteriumtuberculosis complex and said biological sample is selected from thegroup consisting of sputum, bronchial secretion, pleural effusion,gastric juice, blood, spinal fluid, urine, feces, bronchial lavagefluid, and a tissue collected from the bronchus or the lung, lysing thebiological sample with a sample treating agent, subjecting the lysedbiological sample to a heat-treatment carried out at 45° C. to 60° C.for 15 to 60 minutes to induce Mycobacterium tuberculosis complex thatmay be present in the biological sample to extracellularly secrete theMycobacterium tuberculosis complex-specific protein, collecting andpreparing an aliquot from the heat-treated biological sample forimmunological assay, and subjecting the aliquot of the heat-treatedbiological sample to an immunological assay to detect for the presenceof the Mycobacterium tuberculosis complex-specific protein MPB64/MPT64.